Chang-Kang-Fang alleviates diarrhea predominant irritable bowel syndrome (IBS-D) through inhibiting TLR4/NF-κB/NLRP3 pathway.
Chang-Kang-Fang (CKF), originated from traditional Chinese medicine (TCM) formulas, has been utilized to treat diarrhea predominant irritable bowel syndrome (IBS-D) based on clinical experience. However, the underlying mechanism of CKF for treating IBS-D remains unclear and need further clarification.
The objective of this present investigation was to validate the efficacy of CKF on IBS-D model rats and to uncover its potential mechanism for the treatment of IBS-D.
We first established the IBS-D rat model through neonatal maternal separation (NMS) in combination with restraint stress (RS) and the administration of senna decoction via gavage. To confirm the therapeutic effect of CKF on treating IBS-D, abdominal withdrawal reflex (AWR) scores, the quantity of fecal pellets, and the fecal water content (FWC) were measured to evaluate the influence of CKF on visceral hypersensitivity and the severity of diarrhea symptom after the intragastric administration of CKF for 14 days. Subsequently, enzyme linked immunosorbent assay (ELISA) was applied to assess the effect of CKF on neuropeptides substance P (SP) and 5-hydroxytryptamine (5-HT), as well as inflammatory cytokines in serum and in intestinal tissues. Further, colonic pathological changes, the amount of colonic mast cells, and the expression level of occludin in rat colon tissues, were investigated by hematoxylin-eosin (HE) staining, toluidine blue staining, and immunohistochemistry, respectively. To explore the underlying mechanisms, alterations in colonic RNA transcriptomics for the normal, model, and CKF treatment groups were assessed using RNA sequencing (RNA-Seq). Subsequently, quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB), and immunofluorescence (IF) assays were applied to validate the effect of CKF on predicted pathways in vivo and in vitro. In addition, to elucidate the potential active compounds in CKF, 11 representative components found in CKF were selected, and their anti-inflammation potentials were evaluated using LPS-treated RAW264.7 cell models.
CKF treatment significantly reduced the number of fecal pellets, attenuated visceral hypersensitivity, and decreased 5-HT and SP concentrations in serum and colon tissues, along with a reduction in colonic mast cell counts, correlating with improved symptoms in IBS-D rats. Meanwhile, CKF treatment reduced the colonic inflammatory cell infiltration, lowered the levels of IL-6, TNF-α, and IL-1β in serum and colon tissues, and increased the occludin protein expression in colon tissues to improve inflammatory response and colonic barrier function. RNA-Seq, in conjugation with our previous network pharmacology analysis, indicated that CKF might mitigate the symptoms of IBS-D rats by inhibiting the Toll like receptor 4/Nuclear factor kappa-B/NLR family pyrin domain-containing protein 3 (TLR4/NF-κB/NLRP3) pathway, which was confirmed by WB, IF, and qRT-PCR experiments in vivo and in vitro. Furthermore, coptisine, berberine, hyperoside, epicatechin, and gallic acid present in CKF emerged as potential active components for treating IBS-D, as they demonstrated in vitro anti-inflammatory effects.
Our findings demonstrate that CKF effectively improves the symptoms of IBS-D rats, potentially through the inhibition of the TLR4/NF-κB/NLRP3 pathway. Moreover, this study unveils the potential bioactive components in CKF that could be applied in the treatment of IBS-D.
Zhang S
，Tian D
，Xia Z
，Yang F
，Chen Y
，Yao Z
，He Y
，Miao X
，Zhou G
，Yao X
，Tang J
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WenTongGanPi decoction alleviates diarrhea-predominant irritable bowel syndrome by improving intestinal barrier.
WenTongGanPi Decoction (WTGPD) is a representative medical practice of the Fuyang School of Traditional Chinese Medicine (TCM), which originated from the classical Lu's Guizhi method. WTGPD places emphasis on the balance and functionality of yang qi, and is effective in treating TCM symptoms related to liver qi stagnation and spleen yang deficiency. In TCM, diarrhea-predominant irritable bowel syndrome (IBS-D) is often diagnosed as liver depression and spleen deficiency, and the use of WTGPD has shown significant therapeutic effect. However, the underlying mechanism of WTGPD treating IBS-D remains unclear.
To explore the effect and mechanism of WTGPD in the treatment of IBS-D.
An IBS-D model with liver depression and spleen deficiency was constructed by chronic immobilization stress stimulation and sennae folium aqueous gavage. The impact of WTGPD on IBS-D rats was evaluated through measurements of body weight, fecal water content, and abdominal withdrawal reflex (AWR). Intestinal permeability was assessed using hematoxylin-eosin (HE), alcian blue-periodic acid schiff (AB-PAS), immunofluorescence (IF) staining, and quantitative real-time PCR (qRT-PCR). The components of WTGPD were analyzed using UPLC-Q-TOF-MS. The underlying mechanisms were investigated through network pharmacology, transcriptomics sequencing, western blot (WB), molecular docking, and 16S rRNA sequencing.
WTGPD treatment effectively alleviated diarrhea and abnormal pain in IBS-D rats (P < 0.05). It enhanced the intestinal barrier function by improving colonic structure and increasing the expression of tight junction proteins (P < 0.05). A total of 155 components were identified in WTGPD. Both network pharmacology and transcriptomics sequencing analysis highlighted MAPK as the key signaling pathway in WTGPD's anti-IBS-D effect. The WB results showed a significant decrease in p-p38, p-ERK and p-JNK expression after WTGPD treatment (P < 0.0001). Guanosine, adenosine and hesperetin in WTGPD may be involved in regulating the phosphorylation of p38, ERK and JNK. Additionally, WTGPD significantly enhanced microbial diversity and increased the production of colonic valeric acid in IBS-D rats (P < 0.01).
In conclusion, our findings suggest that WTGPD can effectively alleviate IBS-D and improve intestinal barrier likely via inhibiting MAPK signal pathway and improving micobial dysbiosis.
Li Y
，Chen Y
，Liao Z
，Liu Y
，Liu C
，Yang W
，Bai J
，Huang X
，Hao Y
，Liu S
，Liu Y
... -
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The Effect and Mechanism of Sancao Lichang Decoction on Diarrhea- Predominant Irritable Bowel Syndrome by Regulating Tlr4/Myd88/Nf-Κb Pathway.
To evaluate the effect of Sancao Lichang decoction as traditional Chinese medicine on diarrhea-predominant irritable bowel syndrome (IBS-D) and TLR4/MyD88/NF-κB pathway.
Traditional Chinese medicine has made significant progress in preventing and treating irritable bowel syndrome, and its efficacy has been validated by clinical practice. Sancao Lichang decoction is an empirical prescription developed by professor Tang Decai that has been used for many years to treat chronic diarrhoea with good curative effec. Still, its mechanism of action on IBS-D is unknown.
The study sample of Fifty SD rats was randomly divided into a blank group, model group, low-dose group, medium-dose group, and high-dose group (n = 10). The IBS-D rat models were established by restraining stress method and acetic acid enema. After different treatments, defecation frequency, fecal water content (FWC), serum IL-6 and TNF-α contents, and protein level of TLR4/MyD88/NF-κB in colon tissues were detected separately.
The indexes of rats in each group were significantly different. The increase in body weight in the medium-dose and high-dose groups was significantly higher than that in the model group (p < 0.05). Compared with the model group, the medium and high dose groups had lower diarrhea frequency, FWC, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) (p < 0.05). The expression levels of TLR4, MyD88, and NF-κB protein in the colon of the three groups treated with Sancao-Lichan decoction were significantly lower than those in the model group (p < 0.01). After different treatments, the colonic mucosa of rats in each group was stained with HE, which proved that the structural damage of colonic mucosa was improved after treatment with Sancao Lichang decoction, and the improvement effect was dose-dependent.
Sancao Lichang decoction may reduce IBS-D by inhibiting TLR4/MyD88/NF-κB pathway, inhibiting the inflammatory response, and improving intestinal mucosal barrier function.
Zhang P
，Ma Y
，Wang Z
，Tang D
... -
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Atractylodes oil alleviates diarrhea-predominant irritable bowel syndrome by regulating intestinal inflammation and intestinal barrier via SCF/c-kit and MLCK/MLC2 pathways.
Atractylodes lancea (Thunb.) DC. is a widely used traditional herb that is well known for treating spleen deficiency and diarrhea. According to traditional Chinese medicine (TCM) theory, diarrhea-predominant irritable bowel syndrome (IBS-D) is caused by cold and dampness, resulting in diarrhea and abdominal pain. Nevertheless, the effect and mechanism of Atractylodes on IBS-D are still unclear.
This study was designed to confirm the therapeutic effect of Atractylodes lanceolata oil (AO) in a rat model of IBS-D, and to determine the mechanisms by which AO protects against the disease.
The chemical components in AO were determined using gas chromatography-mass spectrometry (GC-MS). The expression levels of 5-hydroxytryptamine (5-HT), vasoactive intestinal peptide (VIP), and surfactant protein (SP) in serum and colon tissue were measured using enzyme-linked immunosorbent assay (ELISA). Reverse transcription-polymerase chain reaction (RT-PCR), western blotting (WB), immunohistochemistry (IHC), and immunofluorescence (IF) were used to elucidate the mechanism of action of AO toward inflammation and the intestinal barrier in a rat model of IBS-D.
The 15 chemical substances of the highest concentration in AO were identified using GC-MS. AO was effective against IBS-D in the rat model, in terms of increased body weight, diarrhea grade score, levels of interleukin-10 (IL-10), aquaporin 3 (AQP3), and aquaporin 8 (AQP8), and reduced fecal moisture content, levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), 5-HT, VIP, and SP, while also reducing intestinal injury, as observed using hematoxylin-eosin (HE) staining. In addition, the results indicated that AO increased the mRNA and protein expression levels of stem cell factor (SCF) and c-kit and enhanced the levels of zonula occludens-1 (ZO-1) and occludin, as well as decreased the levels of myosin light chain kinase (MLCK) and inhibited the phosphorylation of myosin light chain 2 (p-MLC2).
AO was found to be efficacious in the rat model of IBS-D. AO inhibited the SCF/c-kit pathway, thereby reducing inflammation and protecting against intestinal barrier damage via the MLCK/MLC2 pathway.
Xie Y
，Zhan X
，Tu J
，Xu K
，Sun X
，Liu C
，Ke C
，Cao G
，Zhou Z
，Liu Y
... -
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